This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-112: Molecular and Cellular Dynamics Technologies. A central problem in biological research is to understand how the genome is interpreted to determine the large diversity of organismal form and function. In addition, it is critical to identify misregulations that can occur during the interpretation of the genetic information in order to dissect and prevent the development of human diseases. While it has been well established that many important steps in the regulation of gene expression occur at the level of transcription, it has become increasingly clear that posttranscriptional processing is also essential for the precise control of gene expression in eukaryotic cells. Examples include the discovery of RNA-mediated silencing pathways that control mRNA translation or decay and play critical roles during organismal development or during the development of diseases such as cancer. However, in contrast to our excellent understanding of transcriptional control mechanisms, very little is still known about post-transcriptional regulatory programs. This is in part due to the lack of technologies that would enable us to analyze post-transcriptional control steps in a dynamic and non-invasive fashion. In this application, we will address this challenge and propose the development and application of novel labeling methods that will allow us to measure on a system-wide level the turnover of individual RNAs and proteins with high sensitivity and without perturbation of the cellular environment. For the first time, this will allow us to integrate changes that occur in transcription with measurements of mRNA stability, translation and protein turnover. Together, this will allow us to develop a comprehensive overview of the regulation of gene expression under various growth conditions and in different cell types. In the long run, my laboratory will explore new paradigms that emerge from these studies and will elucidate the molecular mechanism underlying novel regulatory responses. These studies will provide critical insight into the fundamental principles that determine the cellular dynamics of gene expression patterns and help us to understand defects that occur in human diseases. PUBLIC HEALTH RELEVANCE: In all eukaryotes, the tight regulation of gene expression is essential for cells to control proliferation, differentiation or development, and errors in any step of the gene expression program can have severe consequences leading to cell death or disease. A critical step in the control of gene expression is the control of mRNA and protein abundance by the regulation of their decay. Therefore, a better understanding of the regulation of mRNA and protein turnover is essential both for the understanding of fundamental cellular processes and the development of novel therapies for human diseases.